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Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 August 2002, Vol. 100, No. 4, pp. 1502-1504 BRIEF REPORT Native human blood dendritic cells as potent effectors in antibody-dependent cellular cytotoxicity Marc Schmitz, Senming Zhao, Knut Schäkel, Martin Bornhäuser, Detlef Ockert, and Ernst Peter Rieber From the Institute of Immunology, Department of Medicine I, and Department of Surgery, Medical Faculty, Technical University of Dresden, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References Functional studies on native human dendritic cells (DCs) are hampered by technical difficulties in preparing fresh DCs. Recently, with the help of the monoclonal antibody M-DC8, we succeeded in isolating a major subpopulation of human blood DCs by a one-step immunomagnetic separation procedure. These cells strongly express FcRIII (CD16) and FcRII (CD32) and are quite efficient in the antigen-specific activation of naive T cells. Because some Fc receptor-bearing cell types are known as effector cells in antibody-dependent cellular cytotoxicity (ADCC), we investigated whether M-DC8+ DCs are capable of effectuating ADCC. In this report we show that freshly prepared M-DC8+ DCs efficiently mediate tumor-directed ADCC and that both types of Fc receptors as well as tumor necrosis factor  essentially contribute to the cytotoxic activity. The results provide evidence that, in addition to their pivotal role in primary T-cell activation, a subset of blood DCs displays efficient cytotoxicity in ADCC. (Blood. 2002;100:1502-1504) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Dendritic cells (DCs) are professional antigen-presenting cells1-3 that are widely used in the experimental immunotherapy of cancer.4 Although in these approaches DCs mediate their antitumor effect by stimulating tumor-specific T lymphocytes, recent observations suggest that DCs themselves have multiple effector cell functions in antitumor reactivity. Thus, DCs were shown to inhibit growth and to induce apoptosis of tumor cell lines in vitro.5,6 In addition, rat spleen DCs expressing natural killer (NK) cell receptor protein 1 were found to lyse tumor cells.7 Recently, we defined a novel major subset of human blood DCs by the use of the monoclonal antibody M-DC8, which recognizes a surface structure selectively expressed on these cells.8 M-DC8+ DCs, which account for 1% to 2% of peripheral blood mononuclear cells (PBMCs) strongly express major histocompatibility complex class II molecules and costimulatory molecules such as CD86. In addition, they efficiently activate neoantigen-specific CD4+ T cells and tumor peptide-specific CD8+ cytotoxic T cells.8 Two blood DC populations, DC1 and DC2, have previously been defined based on their phenotypes (CD11c+/CD123dim and CD11c/CD123high, respectively).3 M-DC8+ DCs, like DC1 cells, are CD11c+/CD123dim yet differ from DC1 and DC2 subsets by the strong expression of FcRIII (CD16) in addition to the M-DC8 antigen. Prompted by the crucial role of FcRIII (CD16) in antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells,9 we investigated whether M-DC8+ DCs are capable of effecting ADCC. So far, reports have demonstrated that human DCs are unable to efficiently mediate ADCC.10,11 Here, we describe that M-DC8+ DCs are able to effectuate tumor-directed ADCC, which is critically dependent on the engagement of FcRIII (CD16) and FcRII (CD32) as well as on the production of tumor necrosis factor  (TNF-).     Study design Top Abstract Introduction Study design Results and discussion References Cell lines and antibodies Colorectal adenocarcinoma cell line COLO 205 and breast cancer cell line SK-BR-3 (American Type Culture Collection, Manassas, VA) were cultured according to the provider's instructions. The following murine monoclonal antibodies were used: 17-1A antibody (IgG2a, Glaxo Wellcome, Bad Oldesloe, Germany) recognizing the epithelial cellular adhesion molecule, isotype-matched control antibody (Becton Dickinson, Heidelberg, Germany), anti-FcRIII (CD16) antibody (clone 3G8, IgG1, Becton Dickinson), anti-FcRII (CD32) antibody (clone AT10, IgG1, Biozol, Eching, Germany), and anti-TNF- antibody (IgG2a, Sigma-Aldrich, Taufkirchen, Germany). The purified isotype-matched control anti-CD3 antibody M-T301 (IgG1)12 was generated in our laboratory. Furthermore, the humanized antibodies Herceptin, directed against the human epidermal growth factor receptor-2, and the isotype-matched control antibody, rituximab, directed against CD20 (IgG1, both from Hoffmann-La Roche, Grenzach-Wyhlen, Germany), were used. Isolation of effector cells Blood samples were obtained from healthy donors with informed consent. Isolation of M-DC8+ DCs was performed as described.8 Briefly, PBMCs were prepared by Ficoll-Hypaque (Biochrom, Berlin, Germany) density centrifugation and incubated for 15 minutes at 4°C with M-DC8 hybridoma supernatant containing 10 µg/mL antibody. After washing with phosphate-buffered saline (PBS) 1 × 108 cells were resuspended in 500 µL PBS and labeled with 10 µL rat anti-mouse IgM coupled to paramagnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) for another 15 minutes at 4°C. After washing, cells were sorted on separation columns (Miltenyi Biotec). PBS containing 1% human serum (CC pro, Neustadt, Germany) was used as running and elution buffer. CD14+ monocytes and CD56+ NK cells were isolated from freshly prepared PBMCs using immunomagnetic separation (Miltenyi Biotec) according to the manufacturer's instructions. Chromium release assay Cytotoxic activity of M-DC8+ DCs was determined against COLO 205 and SK-BR-3 cells in a 51Cr release assay. Briefly, tumor cells (1 × 106) were labeled with 100 µCi (3.7 MBq) 51Cr (sodium chromate, NEN, Zaventem, Belgium) for 1 hour at 37°C and then washed 4 times with PBS. Labeled target cells were plated as triplicates in round bottomed 96-well plates at 5 × 103/well and incubated with various numbers of effector cells for 18 hours in the presence of 10 µg/mL 17-1A antibody, Herceptin, or isotype-matched control antibodies. Released 51Cr was determined in a -counter (Wallac, Freiburg, Germany). Maximal and minimal release were measured by treating labeled cells with 1% Nonidet P-40 or medium alone, respectively. The specific cytotoxicity was calculated according to the formula: percent specific lysis = 100 × [(cpm experimental release  cpm spontaneous release)/(cpm maximal release  cpm spontaneous release)]. To inhibit Fc receptor function during ADCC, M-DC8+ DCs were preincubated with various concentrations of anti-FcRIII (CD16), anti-FcRII (CD32), or an isotype-matched control antibody for 1 hour at 4°C, and then added to tumor cells. To block TNF- function in M-DC8+ DC-mediated ADCC, 51Cr release assay was repeated in the presence of a neutralizing anti-TNF- antibody at a concentration of 10 µg/mL. For statistical evaluation, the Student t test was used. P < .05 was considered significant.     Results and discussion Top Abstract Introduction Study design Results and discussion References To determine the capacity of M-DC8+ DCs to mediate ADCC, DCs were isolated at high purity (> 95%) by immunomagnetic separation from freshly drawn blood of healthy donors and were cocultured with COLO 205 cells expressing the epithelial cellular adhesion molecule or SK-BR-3 cells expressing the human epidermal growth factor receptor-2. As targeting antibodies, either the murine 17-1A antibody or the humanized antibody Herceptin was chosen, both of which have already been used in adjuvant immunotherapy of cancer.13,14 In the presence of 17-1A antibody M-DC8+ DCs efficiently lysed COLO 205 cells (Figure 1A), whereas only marginal tumor cell lysis was found in the absence of antibody or in the presence of an isotype-matched control antibody. In addition, DC8+ DCs efficiently lysed SK-BR-3 cells in the presence of Herceptin antibody (Figure 1B). In further experiments, M-DC8+ DCs were compared with monocytes and NK cells that are regarded as important mediators of ADCC.9,15 All effector cell populations were isolated at high purity (> 95%) as determined by fluorescence-activated cell sorter analysis (data not shown). The cytotoxicity of M-DC8+ DCs was similar to that observed with autologous monocytes, whereas NK cells emerged as the most potent mediators of ADCC in this experimental setting (Figure 1C,D). View larger version (35K): [in this window] [in a new window]   Figure 1. M-DC8+ DC-mediated antibody-dependent cytotoxicity toward COLO 205 and SK-BR-3 tumor cells. (A) Freshly isolated M-DC8+ DCs were cocultured in 96-well plates with 51Cr-labeled COLO 205 cells (5 × 103 cells/well) in the presence of 17-1A antibody (10 µg/mL), an isotype-matched control antibody (10 µg/mL), or in the absence of antibody, at an effector-target (E/T) ratio of 40:1. After 18 hours of incubation, chromium release was measured. The results of 2 different donors are presented as mean ± SE of triplicate wells. (B) M-DC8+ DCs were cocultured in 96-well plates with 51Cr-labeled SK-BR-3 cells (5 × 103 cells/well) in the presence of Herceptin antibody (10 µg/mL), an isotype-matched control antibody (10 µg/mL), or in the absence of antibody, at an E/T ratio of 20:1. After 18 hours of incubation, chromium release was measured. The results of 2 different donors are presented as mean ± SE of triplicate wells. (C) 51Cr-labeled COLO 205 cells (5 × 103 cells/well) were incubated either with M-DC8+ DCs, monocytes, or NK cells in the presence or absence of 10 µg/mL 17-1A antibody at different E/T ratios. After 18 hours of incubation, chromium release was determined. Symbols represent means ± SE of results obtained from 4 different donors. For each donor the mean of triplicate determination was taken. (D) 51Cr-labeled SK-BR-3 cells (5 × 103 cells/well) were incubated either with M-DC8+ DCs, monocytes, or NK cells in the presence or absence of 10 µg/mL Herceptin antibody at different E/T ratios. After 18 hours of incubation, chromium release was determined. Symbols represent means ± SE of results obtained from 4 different donors. For each donor the mean of triplicate determination was taken. The crucial role of Fc receptors in ADCC is well documented.16 Monocytes have been shown to mediate ADCC through FcRI (CD64) and FcRII (CD32).17 This ADCC activity can be enhanced by pretreatment of monocytes with interferon .18 In contrast, ADCC mediated by NK cells is dependent mainly on activation via FcRIII (CD16).9 Because freshly isolated M-DC8+ DCs are characterized by the strong expression of FcRIII (CD16) and FcRII (CD32),8 the differential role of these Fc receptors in M-DC8+ DC-mediated ADCC was evaluated. Anti-FcRIII (CD16) antibody and anti-FcRII (CD32) antibody inhibited ADCC mediated by murine (Figure 2A) as well as humanized antibodies (Figure 2B) to a similar degree. Together with the almost complete reduction of ADCC obtained by the combination of both inhibiting antibodies, these data suggest that the antibody-mediated cytotoxic effect is critically dependent on the simultaneous engagement of both Fc receptors. View larger version (19K): [in this window] [in a new window]   Figure 2. Evaluation of mechanisms underlying ADCC mediated by M-DC8+ DCs. (A) Freshly isolated M-DC8+ DCs were preincubated for 1 hour at 4°C with 50 µg/mL (), 100 µg/mL (), or 200 µg/mL () of either an anti-FcRIII (CD16), or an anti-FcRII (CD32), or an isotype-matched control antibody. In addition, M-DC8+ DCs were preincubated with a combination of an anti-FcRIII (CD16) and an anti-FcRII (CD32) antibody, each at 200 µg/mL (). In this experiment an isotype-matched antibody at 400 µg/mL was used as control (). DCs were cocultured either with 51Cr-labeled COLO 205 cells in the presence of 17-1A (10 µg/mL) at an E/T ratio of 40:1 (A) or with 51Cr-labeled SK-BR-3 cells in the presence of Herceptin (10 µg/mL) at an E/T ratio of 10:1 (B). After 18 hours of incubation, chromium release was measured. Columns represent means ± SE of results obtained from 4 different donors. For each donor the mean of triplicate determination was taken. Asterisks indicate a statistically significant difference (P < .05) between the inhibition of ADCC in the presence of anti-FcRIII (CD16) or anti-FcRII (CD32) antibody or a combination of both anti-FcR antibodies and the isotype-matched control antibody. (C) 51Cr-labeled COLO 205 cells were incubated with M-DC8+ DCs at an E/T ratio of 40:1 in the presence of 10 µg/mL 17-1A antibody with or without a neutralizing anti-TNF- antibody at a concentration of 10 µg/mL. After 18 hours of incubation, chromium release was determined. The results of 2 different donors are presented as mean ± SE of triplicate wells. Tumor necrosis factor  is regarded as an important effector molecule of tumor cell killing.19,20 TNF- production by activated DCs is well documented.21 We observed that during ADCC, M-DC8+ DCs secreted TNF- at a concentration surmounting that observed with autologous monocytes (data not shown). To evaluate the contribution of TNF- to the cytotoxic effect of DCs, ADCC was determined in the presence of a neutralizing anti-TNF- antibody. As demonstrated in Figure 2C, tumor-directed ADCC was almost completely abrogated by the anti-TNF- antibody suggesting an important role of TNF- in ADCC mediated by M-DC8+ DCs. In summary, our data provide for the first time evidence that a major subpopulation of circulating human blood DCs efficiently mediates tumor-directed ADCC through a mechanism that is dependent on the engagement of FcRIII (CD16) and FcRII (CD32) as well as on the production of TNF-. Because immature CD1a+ DCs have been shown to infiltrate tumor tissues22 this novel DC effector function may play an important role in natural and therapeutic antitumor reactions.     Acknowledgments The technical assistance of Karin Günther, Bärbel Löbel, and Verona Schwarze is greatly appreciated.     Footnotes Submitted February 20, 2001; accepted April 8, 2002. Supported by the Wilhelm Sander-Stiftung and by the Medical Faculty, Technical University of Dresden. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Ernst Peter Rieber, Institute of Immunology, Medical Faculty, Technical University of Dresden, Fetscherstr 74, 01307 Dresden, Germany; e-mail: rieber{at}rcs.urz.tu-dresden.de.     References Top Abstract Introduction Study design Results and discussion References 1. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood. 1997;90:3245-3287[Free Full Text]. 2. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252[CrossRef][Medline] [Order article via Infotrieve]. 3. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767-811[CrossRef][Medline] [Order article via Infotrieve]. 4. Fong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol. 2000;18:245-273[CrossRef][Medline] [Order article via Infotrieve]. 5. Chapoval AI, Tamada K, Chen L. In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells. Blood. 2000;95:2346-2351[Abstract/Free Full Text]. 6. Fanger NA, Maliszewksi CR, Schooley K, Griffith TS. Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Exp Med. 1999;190:1155-1164[Abstract/Free Full Text]. 7. Josien R, Heslan M, Soulillou J-P, Cuturi M-C. Rat spleen dendritic cells express natural killer cell receptor protein 1 (NKR-P1) and have cytotoxic activity to select targets via a Ca2+-dependent mechanism. J Exp Med. 1997;186:467-472[Abstract/Free Full Text]. 8. Schäkel K, Mayer E, Federle C, Schmitz M, Riethmüller G, Rieber EP. A novel dendritic cell population in human blood: one-step immunomagnetic isolation by a specific mAb (M-DC8) and in vitro priming of cytotoxic T lymphocytes. Eur J Immunol. 1998;28:4084-4093[CrossRef][Medline] [Order article via Infotrieve]. 9. Leibson PJ. Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity. 1997;6:655-661[CrossRef][Medline] [Order article via Infotrieve]. 10. Fanger NA, Voigtlaender D, Liu C, et al. Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor FcRI (CD64) expressed on human blood dendritic cells. J Immunol. 1997;158:3090-3098[Abstract]. 11. Boyer A, Andreu G, Romet-Lemonne J-L, Fridman W-H, Teillaud J-L. Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties. Exp Hematol. 1999;27:751-761[CrossRef][Medline] [Order article via Infotrieve]. 12. Rieber EP. T-cell section report. In: Knapp W,Dörken B,Gilks WR, et al., eds. Leukocyte Typing IV. Oxford United Kingdom: Oxford University Press; 1989:229-249. 13. Riethmüller G, Holz E, Schlimok G, et al. Monoclonal antibody therapy for resected Dukes' C colorectal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol. 1998;16:1788-1794[Abstract]. 14. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER. N Engl J Med. 2001;344:783-792[Abstract/Free Full Text]. 15. Munn DH, Cheung NK. Antibody-dependent antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor: induction of efficient antibody-mediated antitumor cytotoxicity not detected by isotope release assays. J Exp Med. 1989;170:511-526[Abstract/Free Full Text]. 16. Van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today. 1993;14:215-221[CrossRef][Medline] [Order article via Infotrieve]. 17. Graziano RF, Fanger MW. Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells. J Immunol. 1987;139:3536-3541[Abstract]. 18. Van Schie RCAA, Verstraten RGG, Van de Winkel JGJ, Tax WJM, de Mulder PHM. Effect of recombinant interferon-gamma (IFN-gamma) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity: rIFN-gamma decreases inhibition of cytophilic human IgG and changes the cytolytic mechanism. J Immunol. 1992;148:169-176[Abstract]. 19. Feinman R, Hendriksen-DeStefano D, Tsujimoto M, Vilcek J. Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J Immunol. 1987;138:635-640[Abstract]. 20. Pullyblank AM, Guillou PJ, Monson JRT. Interleukin 1 and tumour necrosis factor alpha may be responsible for the lytic mechanism during anti-tumour antibody-dependent cell-mediated cytotoxicity. Br J Cancer. 1995;72:601-606[Medline] [Order article via Infotrieve]. 21. Verhasselt V, Buelens C, Willems F, De Groote D, Haeffner-Cavaillon N, Goldman M. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J Immunol. 1997;158:2919-2925[Abstract]. 22. Bell D, Chomarat P, Broyles D, et al. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J Exp Med. 1999;190:1417-1425[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Z. M. Bamboat, J. A. Stableford, G. Plitas, B. M. Burt, H. M. Nguyen, A. P. Welles, M. Gonen, J. W. Young, and R. P. 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